The GeneRead DNA FFPE Kit enables purification of high-quality genomic DNA (gDNA) from formalin-fixed paraffin embedded (FFPE) tissue samples using an optimized silica spin-column–based protocol. Artificial C>T mutations caused by cytosine deamination are critical in next-generation sequencing (NGS). These artifacts are caused by formalin fixation and aging, and result in sequencing errors. The GeneRead FFPE purification procedure also includes enzymatic removal of these artifacts, while ensuring high yields and purity, making it especially suitable for NGS applications.

The GeneRead DNA FFPE Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

High yields of double-stranded DNA.

Single 10 μm slices of FFPE tissue were used to compare the results of DNA purification using standard and GeneRead protocols for FFPE samples. The wide range of DNA yields seen is due to the variation in the starting material. This is common for DNA purification from FFPE samples.

GeneRead DNA FFPE Kit outperforms a kit from another supplier.

Double-stranded DNA was extracted from single 10 μm slices of FFPE tissue using the GeneRead DNA FFPE Kit and a product from an alternative supplier. Yields were measured by Qubit Fluorometric Quantitation.The GeneRead DNA FFPE Kit delivered up to 4-fold higher yields for the samples tested. A wide range of DNA yields is seen due to variation in the starting material, which is common for DNA extractions from FFPE samples.

Dramatic reduction in artifactual C>T|G>A transitions.

Analysis of liver cancer samples was carried out. The GeneRead DNA FFPE Kit removed over 90% of low-frequency, novel mutations that are most likely to be artifactual in nature.

In FFPE samples, cytosine may become deaminated, leading to the cytosine pairing with adenine as uracil. In sequencing reactions, this will manifest as a C>T|G>A transition.
(Figure adapted from Klug and Cummings, 1997)

Performance

High yields from limited starting material

Advances in library preparation and sequencing technologies have made it attractive to perform high-throughput sequencing on large amounts of biobanked FFPE tissues. These technologies have also lowered the frequency threshold at which sequence mutations can be reliably detected. However, sequencing FFPE samples is associated with various challenges. Yields from FFPE samples may be limited due to the compromised status of the DNA. Additionally, these samples are often irreplaceable and there is a need to get the maximum amount of nucleic acid from the smallest amount of starting material. In addition to yield, artifact suppression becomes critical when sequencing FFPE samples, as the relative frequency of false mutations is increased when starting with limited material. The GeneRead DNA FFPE Kit delivers equal or higher yields of double-stranded DNA compared to a standard FFPE DNA isolation protocol (see figure High yields of double-stranded DNA) from small samples (1 x 10 µm slide). The GeneRead DNA FFPE Kit outperforms a kit from another supplier, delivering up to 4-fold higher yields of double-stranded DNA for the samples tested (see figure GeneRead DNA FFPE Kit outperforms a kit from another supplier).

Efficient reduction of artifactual C>T|G>A transitions

DNA damage caused by formalin fixation and storage is essentially random in nature, with resulting altered sites distributed across the sequence. Only a few copies of the genome will be damaged at any site, leading to a generally low frequency of these artifacts. As low-frequency, novel mutations can be very important, for example in cancer analysis, it is important to distinguish between true and false low-frequency mutations. By examining low-frequency, novel mutations, the number of artifacts in a sample was estimated and the efficacy of the artifact removal process in the GeneRead DNA FFPE protocol was determined. The GeneRead DNA FFPE Kit dramatically reduces low-frequency C>T|G>A transitions while retaining true mutations (see figures Dramatic reduction in artifactual C>T|G>A mutations and Retention of high-frequency C>T|G>A transitions).

Minimizing the risk of false-positive mutations

The removal of artifactual C>T|G>A transitions is especially important when analyzing SNPs associated with cancer. Since deamination mutations occur randomly, they may also be interpreted as cancer-relevant and thereby appear as false-positive SNPs if no artifact removal is employed. The GeneRead DNA FFPE Kit minimizes the risk of false positives as only artifactual mutations are removed (see poster).

Principle

DNA preparation from FFPE tissue for next-generation sequencing applications is associated with several challenges. Yields are often limited due to the precious nature of the sample and the compromised status of the DNA. Additionally, artifacts introduced by fixation and embedding conditions, and due to long-term storage, are most prevalent in sequencing results when starting with limited material. One particular problem is the deamination of cytosine bases to deoxyuracil. This leads to a C-T conversion in sequencing reactions. While the exact mechanism for this is unknown, one explanation is that deamination of cytosine leads to a uracil in that position, which will pair with adenine (see figure Deamination of cytosine leads to false adenine pairing). Upon sequencing, this modified base will be read as a C>T transition. If strand information is not preserved during library construction, either strand may be sequenced, and therefore, the artifact may appear as either a C>T or a G>A transition. Removal of these artifacts is essential, particularly when used for sequencing analysis of cancer samples, as they may otherwise appear as false-positive mutations. Artifact suppression becomes critical when sequencing FFPE samples, as the relative frequency of false mutations is increased when starting with limited material.

The GeneRead DNA FFPE Kit provides a streamlined procedure for efficient purification of high yields of DNA from small amounts of FFPE tissue sections. Additionally, the procedure includes the removal of deaminated cytosine to prevent false results in DNA sequencing.

Procedure

The GeneRead DNA FFPE procedure removes paraffin and reverses formalin cross-links from the DNA sample before it is bound to the QIAamp MinElute column. After heating to remove cross-links, the DNA is accessible for the specific removal of deaminated cytosine residues by the enzyme Uracil-N-Glycosilase (UNG). The optimized reaction mixture provides conditions in which the UNG can specifically remove artificially induced uracils from the DNA obtained from the FFPE sample. After the binding of DNA to the spin column, residual contaminants such as salts are washed away by Buffers AW1 and AW2, and ethanol. Any residual ethanol, which may interfere with subsequent enzymatic reactions, is removed by an additional centrifugation step. DNA is eluted and is now ready to use in next-generation sequencing workflows. Alternatively, it can be stored at –20°C.